Activation of the Luteinizing Hormone β Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH2-terminal Protein Kinase
2000; Elsevier BV; Volume: 275; Issue: 28 Linguagem: Inglês
10.1074/jbc.m910252199
ISSN1083-351X
AutoresTakeshi Yokoi, Masahide Ohmichi, Keiichi Tasaka, Akiko Kimura, Yuki Kanda, Jun Hayakawa, Masahiro Tahara, Koji Hisamoto, Hirohisa Kurachi, Yuji Murata,
Tópico(s)Reproductive Biology and Fertility
ResumoRegulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LβT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca2+, whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca2+. To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone β (LHβ) promoter activity, we transfected the rat LHβ (−156 to +7)-luciferase construct into LβT2 cells. GnRH activated the rat LHβ promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHβ promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHβ promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHβ promoter by GnRH. In addition, GnRH did not induce either the rat LHβ promoter activity in LβT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHβ promoter activity in a c-Jun-dependent mechanism in LβT2 cells. Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LβT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca2+, whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca2+. To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone β (LHβ) promoter activity, we transfected the rat LHβ (−156 to +7)-luciferase construct into LβT2 cells. GnRH activated the rat LHβ promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHβ promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHβ promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHβ promoter by GnRH. In addition, GnRH did not induce either the rat LHβ promoter activity in LβT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHβ promoter activity in a c-Jun-dependent mechanism in LβT2 cells. gonadotropin-releasing hormone gonadotropin-releasing hormone agonist mitogen-activated protein kinase extracellular signal-regulated (protein) kinase c-Jun NH2-terminal protein kinase stress-activated protein kinase a catalytically inactive form of MAPK dominant negative c-Jun myelin basic protein glutathione S-transferase polyacrylamide gel electrophoresis pertussis toxin protein kinase C phorbol 12-myristate 13-acetate 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester mitogen-activated protein kinase/extracellular signal-regulated kinase kinase luteinizing hormone follicle-stimulating hormone cytomegalovirus hemagglutinin GnRH,1 a hypothalamic decapeptide, serves as a key regulator of the reproductive system. GnRH acts on anterior pituitary gonadotropes to stimulate the synthesis and release of the pituitary gonadotropins LH and FSH. The gonadotropins are subunit hormones, each containing noncovalently linked α- and β-subunits (1.Pierce J.G. Parsons T.F. Annu. Rev. Biochem. 1981; 50: 465-495Crossref PubMed Scopus (1901) Google Scholar, 2.Gharib S.D. Wierman M.E. Shupnik M.A. Chin W.W. Endocr. Rev. 1990; 11: 177-199Crossref PubMed Scopus (604) Google Scholar). Within a species, the α-subunits are identical, while the β-subunits differ and confer the physiological specificity of the heterodimeric hormone. Each β-subunit as well as the common α-subunit is encoded by different genes on separate chromosomes. When GnRH binds to its seven-transmembrane receptor (3.Tsutsumi M. Zhou W. Millar R.P. Mellon P.L. Roberts J.L. Flanagan C.A. Dong K. Gillo B. Sealfon S.C. Mol. Endocrinol. 1992; 6: 1163-1169Crossref PubMed Scopus (218) Google Scholar), it induces interaction of the receptor with the heterotrimeric Gqprotein, which leads to activation of phospholipase C and formation of inositol 1,4,5-triphosphate and diacylglycerol, leading to elevation of intracellular Ca2+ and activation of protein kinase C (PKC) (4.Horn F. Bilezikjian L.M. Perrin M.H. Bosma M.M. Windle J.J. Huber K.S. Blount A.L. Hille B. Vale W. Mellon P.L. Mol. Endocrinol. 1991; 5: 347-355Crossref PubMed Scopus (144) Google Scholar, 5.Huckle W.R. Conn P.M. Endocrine Rev. 1988; 9: 387-395Crossref PubMed Scopus (141) Google Scholar, 6.Reinhart J. Mertz L.M. Catt K.J. J. Biol. Chem. 1992; 267: 21281-21284Abstract Full Text PDF PubMed Google Scholar). Intracellular transmission of extracellular signals is mediated in large part by several groups of sequentially activated protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) cascades. In growth factor signaling, the key elucidated MAPK cascade is the extracellular signal-regulated kinase (ERK). Recent evidence indicates that many G protein-coupled receptors can activate the ERK cascade (7.Ohmichi M. Sawada T. Kanda Y. Koike K. Hirota K. Miyake A. Saltiel A.R. J. Biol. Chem. 1994; 269: 3783-3788Abstract Full Text PDF PubMed Google Scholar, 8.Ohmichi M. Koike K. Nohara A. Kanda Y. Sakamoto Y. Zhang Z.X. Hirota K. Miyake A. Endocrinology. 1995; 136: 2082-2087Crossref PubMed Google Scholar, 9.Ohmichi M. Koike K. Kimura A. Masuhara K. Ikegami H. Ikebuchi Y. Kanzaki T. Touhara K. Sakaue M. Kobayashi Y. Akabane M. Miyake A. Murata Y. Endocrinology. 1997; 138 (3111): 3130Crossref Scopus (44) Google Scholar, 10.Sawada T. Ohmichi M. Koike K. Kanda Y. Kimura A. Masuhara K. Ikegami H. Inoue M. Miyake A. Murata Y. Endocrinology. 1997; 138: 5275-5281Crossref PubMed Scopus (13) Google Scholar, 11.Kimura A. Ohmichi M. Takeda T. Kurachi H. Ikegami H. Koike K. Masuhara K. 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Cell. 1994; 76: 1025-1027Abstract Full Text PDF PubMed Scopus (2954) Google Scholar) cascade, which is known to be activated in response to cellular stresses such as apoptosis (18.Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2413) Google Scholar, 20.Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1446) Google Scholar). ERK, JNK, and p38 (21.Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (997) Google Scholar) constitute the MAPK family. Recent data suggest that GnRH is capable of activating JNK (22.Levi N.L. Hanoch T. Benard O. Rozenblat M. Harris D. Reiss N. Naor Z. Seger R. Mol. Endocrinology. 1998; 12: 815-824Crossref PubMed Google Scholar) and p38 (23.Roberson M.S. Zhang T. Li H.L. Mulvaney J.M. Endocrinology. 1999; 140: 1310-1318Crossref PubMed Google Scholar) in the αT3–1 gonadotrope cell line. It was reported that GnRH induction and basal control of the α-subunit gene seem to occur through the PKC/ERK pathway, while induction of the LHβ gene is dependent on calcium influx in the αT3–1 gonadotrope cell line, suggesting the differential stimulation of transcription of rat LH subunit genes by GnRH (24.Weck L. Fallest P.C. Pitt L.K. Shupnik M.A. Mol. Endocrinol. 1998; 12: 451-457Crossref PubMed Google Scholar). However, the αT3–1 gonadotrope cell line does not express the LHβ gene. Mellon and co-workers (25.Mellon P.L. Windle J.J. Weiner R.J. Rec. Prog. Horm. Res. 1991; 47: 69-96PubMed Google Scholar), using targeted oncogenesis in transgenic mice, have recently generated an immortal gonadotrope cell line (LβT2). The cells of this line express the mRNA of GnRH receptor and of both the α- and β-subunits of LH (26.Alarid E.T. Windle J.J. Whyte D.B. Mellon P.L. Developmemt. 1996; 122: 3319-3329Crossref PubMed Google Scholar, 27.Turgeon J. Kimura Y. Waring D.W. Mellon P.L. Mol. Endocrinol. 1996; 10: 439-450Crossref PubMed Google Scholar). Taken together, these facts led us to examine whether GnRH stimulates the activity of ERK and/or JNK, and whether the respective cascades play a role in the transcriptional activation of the rat LHβ gene in LβT2 cells. Phorbol 12-myristate 13-acetate (PMA) and myelin basic protein were purchased from Sigma. Bisindolylmaleimide (GF 109203X) was purchased from Calbiochem (Laufelfingen, Switzerland). GnRH was obtained from Peninsula Laboratories (Belmont, CA). GnRH agonist, [d-Leu6,Por9-NHEt]leuprolide, was a gift from Takeda Chemical Industries (Japan). ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech. [γ-32P]ATP (3000 Ci/mmol) was obtained from NEN Life Science Products. Erk1 rabbit polyclonal anti-ERK antibody, anti-Myc antibody, and anti-HA antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059 and the SAPK/JNK assay kit, including NH2-terminal c-Jun fusion protein bound to glutathione-Sepharose beads and a phosphospecific c-Jun antibody (Ser63), were obtained from New England Biolabs (Beverly, MA). LβT2 cells (26.Alarid E.T. Windle J.J. Whyte D.B. Mellon P.L. Developmemt. 1996; 122: 3319-3329Crossref PubMed Google Scholar) were generously provided by P. Mellon (La Jolla, CA). Cells were cultured at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a water-saturated atmosphere of 95% O2 and 5% CO2. The wild type −156 to +7 LHβ promoter construct, cloned upstream of luciferase in PL(KS)b-Luc vector, was a kind gift from Dr. Y. Sadovsky (Washington University School of Medicine, St. Louis, MO) (28.Dorn C. Ou Q. Svaren J. Crawford P.A. Sadovsky Y. J. Biol. Chem. 1999; 274: 13870-13876Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The plasmid pLNCX-MAPK (K→M) (29.Keech C.A. Gutierrez-Hartmann A. Mol. Cell. Endocrinol. 1991; 78: 55-60Crossref PubMed Scopus (20) Google Scholar) was a kind gift from Dr. A. Gutierrez-Hartmann (University of Colorado Health Sciences Center, Denver, CO). Plasmid encoding the dominant negative form of Ets-2 (30.Aperlo C. Pognonec P. Stanley R. Boulukos K.E. Mol. Cell. Biol. 1996; 16: 6851-6858Crossref PubMed Scopus (37) Google Scholar) was a kind gift from Dr. K. E. Boulukos (Center de Biochimie, Faculté des Sciences, Nice, France). pAPr-etsZ, encoding the consensus DNA-binding domain of Ets-2, was a kind gift from Dr. M. Ostrowski (Ohio State University, Columbus, OH) (31.Langer S.J. Bortner D.M. Roussel M.F. Sherr C.J. Ostrowski M.C. Mol. Cell. Biol. 1992; 12: 5355-5362Crossref PubMed Scopus (136) Google Scholar). 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The dominant negative c-Jun (dnJun) expression plasmid pLHCc-JUN (S63A, S73A) was constructed as described previously (32.Potapova O. Fakrai H. Baird S. Mercola D. Cancer Res. 1996; 56: 2800-2806Google Scholar). LβT2 cells were transfected for 12 h in six-well tissue culture plates with 2 μg of pLHCdnc-JUN (S63A, S73A) using LipofectAMINE Plus (Life Technologies, Inc.) (36.Hayakawa J. Ohmichi M. Kurachi H. Ikegami H. Kimura A. Matsuoka T. Jukihara H. Mercola D Murata Y. J. Biol. Chem. 1999; 274: 31648-31654Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Clone selection was performed by adding hygromycin to the medium at 200 μg/ml final concentration 2 days after the transfection. After 3 weeks, several clones were isolated using cloning rings. Selected clones were then maintained in medium supplemented with hygromycin (100 μg/ml), and only low passage cells (p < 10) were used for the experiments described here. Cells were incubated overnight in the absence of serum and then treated with various substances. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 μm sodium orthovanadate, 100 mm NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride) (37.Ohmichi M. Matuoka K. Takenawa T. Saltiel A.R. J. Biol. Chem. 1994; 269: 1143-1148Abstract Full Text PDF PubMed Google Scholar). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent. Erk1 rabbit polyclonal antibody was bound to protein A-Sepharose beads, and 300 μg of protein from the lysate samples was immunoprecipitated at 4 °C for 2 h. The immunoprecipitated products were washed once in HNTG buffer, twice in 0.5 m LiCl, 0.1 mTris, pH 8.0, and once in kinase assay buffer (25 mm HEPES, pH 7.2–7.4, 10 mm MgCl2, 10 mmMnCl2, and 1 mm dithiothreitol), and samples were resuspended in 30 μl of kinase assay buffer containing 10 μg of myelin basic protein and 40 μm[γ-32P]ATP (1 μCi) as described previously (16.Sundaresan S. Colin I.M. Pestell R.G. Jameson J.L. Endocrinology. 1996; 137: 304-311Crossref PubMed Scopus (142) Google Scholar). The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (38.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206977) Google Scholar). Reaction products were resolved by 15% SDS-PAGE. This activity was assayed as described (9.Ohmichi M. Koike K. Kimura A. Masuhara K. Ikegami H. Ikebuchi Y. Kanzaki T. Touhara K. Sakaue M. Kobayashi Y. Akabane M. Miyake A. Murata Y. Endocrinology. 1997; 138 (3111): 3130Crossref Scopus (44) Google Scholar, 10.Sawada T. Ohmichi M. Koike K. Kanda Y. Kimura A. Masuhara K. Ikegami H. Inoue M. Miyake A. Murata Y. Endocrinology. 1997; 138: 5275-5281Crossref PubMed Scopus (13) Google Scholar, 39.Kimura A. Ohmichi M. Kurachi H. Ikegami H. Hayakawa J. Tasaka K. Kanada Y. Jikihara H. Matsuura N. Murata Y. Cancer Res. 1999; 59: 5133-5142PubMed Google Scholar). Briefly, LβT2 cells cultured in 100-mm dishes were transfected with Myc-tagged p42MAPK expression plasmid (1 μg of pEXV-Erk2-tag) in combination with 9 μg of pLNCX, pLNCX-MAPK (K→M), pcDL-SRα, or pcDL-SRα-SAPK-VPF using LipofectAMINE Plus. At 72 h after transfection, serum-deprived cells were incubated with 1 μm GnRHa for 5 min, and the expressed Myc-tagged p42MAPK was immunoprecipitated with 1 μg of antibody 9E10. The ERK activity in the immunoprecipitate was measured as described above. JNK activity was precipitated from 250 μg of whole cell lysates by incubation with 2 μg of GST-c-Jun-(1–89) fusion protein/GSH-Sepharose beads for 18 h at 4 °C (New England Biolabs; Ref. 19.Derijard B. Hibi M. Wu L.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1027Abstract Full Text PDF PubMed Scopus (2954) Google Scholar). c-Jun-(1–89) contains a high affinity binding site for JNK close to the NH2 terminus; this site contains two phosphorylation sites at Ser63 and Ser73. The beads were washed and resuspended in 50 μl of kinase buffer containing 100 μm ATP for 30 min at 30 °C as described (39.Kimura A. Ohmichi M. Kurachi H. Ikegami H. Hayakawa J. Tasaka K. Kanada Y. Jikihara H. Matsuura N. Murata Y. Cancer Res. 1999; 59: 5133-5142PubMed Google Scholar). The solid-phase kinase reaction was terminated by addition of Laemmli sample buffer, and phosphorylation of GST-c-Jun on Ser63 was examined after SDS-PAGE and immunoblotting with anti-phospho(Ser63) c-Jun antibody. LβT2 cells cultured in 100 mm dishes were transfected with HA-tagged wild type SAPK/JNK expression plasmid (1 μg of pcDL-SRα-wt-SAPK) or HA-tagged dominant negative SAPK/JNK expression plasmid (1 μg of pcDL-SRα-SAPK-VPF) using LipofectAMINE Plus. At 72 h after transfection, serum-deprived cells were incubated with 1 μm GnRHa for 30 min, and cell lysates were immunoprecipitated with anti-HA antibody. The expressed HA-tagged wild type SAPK/JNK or dominant negative SAPK/JNK was eluted with 1% SDS, and the JNK activity was measured as described above. LβT2 cells cultured in 24-well plates were transfected with the rat −156 to +7 LHβ-luciferase construct and CMV-β-galactosidase plasmid (to normalize for cell viability and transfection efficiency) in combination with the indicated plasmids using LipofectAMINE Plus. At 48 h after transfection, serum-deprived cells were incubated with 100 nm GnRH for the indicated times. In some of the experiments, cells were treated with 20 μm PD98059 for 15 min before the addition of 100 nm GnRH. Cell extracts were prepared by lysing the cells with three sequential freeze-thaw cycles in a buffer containing 100 mm potassium phosphate, pH 7.8, and 10 mm dithiothreitol. Vigorous vortexing was used to enhance cell lysis. Unlysed cells and insoluble material were pelleted at 10,000 rpm for 10 min at 4 °C. The supernatant volume was measured, and aliquots of the supernatant were used in the subsequent luciferase and β-galactosidase assays. Luciferase was assayed as described previously (40.Kimura A. Ohmichi M. Tasaka K. Kanda Y. Ikegami H. Hayakawa J. Hisamoto K. Morishige K. Hinuma S. Kurachi H. Murata Y. J. Biol. Chem. 2000; 275: 3667-3674Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Briefly, the luciferase assay mixture contained 100 mm KPO4, pH 7.8, 1 mm dithiothreitol, 3.7 mmMgSO4, 530 μm ATP, and 470 μmluciferin plus 20 μl of cell extract in a final volume of 100 μl. Luciferin was added just before measuring light units, which were measured in duplicate during the first 40 s of the reaction at 25 °C in a luminometer (41.de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2478) Google Scholar). β-Galactosidase was assayed as described previously (40.Kimura A. Ohmichi M. Tasaka K. Kanda Y. Ikegami H. Hayakawa J. Hisamoto K. Morishige K. Hinuma S. Kurachi H. Murata Y. J. Biol. Chem. 2000; 275: 3667-3674Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The β-galactosidase buffer contained 60 mm sodium phosphate, pH 7.5, 1 mm MgCl2, 0.80 mg/mlO-nitrophenyl-β-δ−galactopyranoside, and 40 mm β-mercaptoethanol. A standard curve for 100 microunits to 2 milliunits of β-galactosidase was made with each assay. A 30-μl aliquot of cell extract was incubated with assay buffer until color developed (30–120 min), and the reaction was then stopped by adding Na2CO3 to a final concentration of 625 mm. Absorbance was then read at 405 nm. Luciferase light units were normalized relative to the activity of β-galactosidase. The control value was set at 1 and the data expressed as -fold stimulation relative to control. Data are expressed as the mean ± S.E. Statistical analysis was performed by Student'st test, and p < 0.01 was considered significant. Data are expressed as the mean ± S.E. To evaluate whether ERK is activated by GnRH in LβT2 cells, cultured cells were exposed to GnRHa for the indicated times (Fig.1 A). Cell lysates were immunoprecipitated with anti-ERK antibody and examined for ERK activity by assaying the incorporation of 32P into MBP. The GnRHa-dependent increase in ERK activity reached a plateau from 5 min through 10 min and rapidly declined thereafter. We next examined the effect of GnRH on the activation of JNK, which is a member of the MAP kinase family. Cultured cells were exposed to GnRHa for the indicated times and cell lysates were incubated with GST-c-Jun fusion protein, followed by precipitation and Western analysis using anti-phospho-c-Jun antibody (Fig. 1 B). The activation of JNK by GnRHa in LβT2 cells was detectable at 5 min, reached a broad plateau from 30 min through 3 h, and declined thereafter. These results indicate that JNK activation by GnRHa was slower than ERK activation. We also found that GnRH activated both ERK and JNK, and that the time courses for ERK and JNK activation by GnRH were similar to that of GnRHa (data not shown). We compared the mechanisms of ERK and JNK activation induced by GnRH. It has been shown that the receptor for GnRH (3.Tsutsumi M. Zhou W. Millar R.P. Mellon P.L. Roberts J.L. Flanagan C.A. Dong K. Gillo B. Sealfon S.C. Mol. Endocrinol. 1992; 6: 1163-1169Crossref PubMed Scopus (218) Google Scholar, 4.Horn F. Bilezikjian L.M. Perrin M.H. Bosma M.M. Windle J.J. Huber K.S. Blount A.L. Hille B. Vale W. Mellon P.L. Mol. Endocrinol. 1991; 5: 347-355Crossref PubMed Scopus (144) Google Scholar, 5.Huckle W.R. Conn P.M. Endocrine Rev. 1988; 9: 387-395Crossref PubMed Scopus (141) Google Scholar, 6.Reinhart J. Mertz L.M. Catt K.J. J. Biol. Chem. 1992; 267: 21281-21284Abstract Full Text PDF PubMed Google Scholar) is a member of the superfamily of G protein-coupled receptors. To determine what type of G protein is coupled to the GnRH receptor, we pretreated LβT2 cells with 100 ng/ml pertussis toxin (PTX) for 4 h in order to inactivate Gi and Go proteins, and then treated the cells with 1 μm GnRHa for 5 min (Fig. 2 A) or 30 min (Fig.2 B, upper panel). Whereas PTX clearly caused a decrease in GnRHa-induced ERK activation (Fig. 2 A,lane 7), PTX did not have a detectable effect on GnRHa-induced JNK (Fig. 2 B, upper panel, lane 7) activation. Thus, although PTX-sensitive G proteins are partly involved in the effect of GnRHa on ERK activity, as previously reported (42.Sim PJ Wolbers WB Mitchell R Mol. Cell. Endocrinol. 1995; 112: 257-263Crossref PubMed Scopus (43) Google Scholar), PTX-sensitive G proteins are not involved in the effect of GnRHa on JNK activity, as was also previously reported (22.Levi N.L. Hanoch T. Benard O. Rozenblat M. Harris D. Reiss N. Naor Z. Seger R. Mol. Endocrinology. 1998; 12: 815-824Crossref PubMed Google Scholar). Many G protein-linked receptors can mediate stimulation of ERK activity via the phospholipase C-dependent activation of PKC (43.Pang L. Decker S.J. Saltiel A.R. Biochem. J. 1993; 289: 283-287Crossref PubMed Scopus (71) Google Scholar, 44.Bogoyevitch M.A. Glennon P.E. Sugden P.H. FEBS Lett. 1993; 317: 271-275Crossref PubMed Scopus (166) Google Scholar, 45.Wang Y. Simomson M.S. Pouyssegur J. Dunn M.J. Biochem. J. 1992; 287: 589-594Crossref PubMed Scopus (161) Google Scholar, 46.Duff J.L. Berk B.C. Corson M.A. Biochem. Biophys. Res. Commun. 1992; 188: 257-264Crossref PubMed Scopus (197) Google Scholar). Activation of ERK (16.Sundaresan S. Colin I.M. Pestell R.G. Jameson J.L. Endocrinology. 1996; 137: 304-311Crossref PubMed Scopus (142) Google Scholar, 17.Reiss N. Llevi L.N. Shacham S. Harris D. Seger R. Naor Z. Endocrinology. 1996; 138: 1673-1682Crossref Scopus (125) Google Scholar) or JNK (22.Levi N.L. Hanoch T. Benard O. Rozenblat M. Harris D. Reiss N. Naor Z. Seger R. Mol. Endocrinology. 1998; 12: 815-824Crossref PubMed Google Scholar) by GnRH requires PKC in αT3–1 cells. Therefore, the role of PKC in GnRH-induced ERK (Fig.2 A) or JNK (Fig. 2 B) activation in LβT2 cells was examined. Exposure of LβT2 cells to PMA caused stimulation of ERK activity (Fig. 2 A, lane 1). However, the ability of PMA to induce the activation of ERK does not necessarily mean that the PKC pathway is involved in GnRHa-induced ERK activation, as has been shown in the case of norepinephrine-induced ERK activation in both adipocytes (47.Shimizu Y. Tanishita T. Minokoshi Y. Shimazu T. Endocrinology. 1997; 138: 248-253Crossref PubMed Scopus (41) Google Scholar) and GT-1 GnRH neuronal cell lines (10.Sawada T. Ohmichi M. Koike K. Kanda Y. Kimura A. Masuhara K. Ikegami H. Inoue M. Miyake A. Murata Y. Endocrinology. 1997; 138: 5275-5281Crossref PubMed Scopus (13) Google Scholar). Whether PKC is indeed involved in GnRH signaling was determined using PKC depletion. Pretreatment with 1 μm PMA for 16 h to deplete most PKC isoforms completely abolished the GnRHa-induced ERK activation (Fig. 2 A, lane 6). On the other hand, treatment with 1 μm PMA for 5 min (Fig.2 B, upper panel, lane 1) or for 30 min (Fig. 2 B, lower panel, lane 5) did not induce JNK activation. Moreover, neither pretreatment with 1 μm PMA for 16 h (Fig. 2 B, upper panel,lane 6) nor pretreatment with the selective PKC inhibitor GF 109203X (48.Mischak H. Goodnight J. Kolch W. Martiny-Baron G. Schaechte C. Kazanietz M.G. Blumberg P.M. Pierce J.H. Mushinski J.F. J. Biol. Chem. 1993; 268: 6090-6096Abstract Full Text PDF PubMed Google Scholar) at 10 μm (Fig. 2 B,lower panel, lane 4) had any effect on the GnRHa-induced JNK activation. These results suggest that activation of ERK by GnRH was mediated by PKC, whereas activation of JNK by GnRH was not mediated by PKC. It has been reported that elevated Ca2+ is necessary for GnRH-induced ERK activation in αT3–1 cells (16.Sundaresan S. Colin I.M. Pestell R.G. Jameson J.L. Endocrinology. 1996; 137: 304-311Crossref PubMed Scopus (142) Google Scholar, 17.Reiss N. Llevi L.N. Shacham S. Harris D. Seger R. Naor Z. Endocrinology. 1996; 138: 1673-1682Crossref Scopus (125) Google Scholar). We therefore evaluated the role of extracellular and intracellular Ca2+ in the GnRH- induced ERK (Fig. 3 A) and JNK (Fig.3 B) activation in LβT2 cells. Elimination of extracellular Ca2+ by treatment with 3 mm EGTA for 1 min or with 1 μm nifedipine for 10 min clearly attenuated GnRHa-induced ERK activation (Fig. 3 A, lanes 3 and 5), indicating that Ca2+ influx is required for GnRHa-induced ERK activation. Moreover, treatment with either 50 μm1,2-bis(o-aminophenoxy)ethane-N,N,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) for 20 min to eliminate intracellular Ca2+ (Fig. 3 A, lane 6) or 3 mm EGTA for 15 min to eliminate extracellular and intracellular Ca2+ (49.Chao T.S.O. Byron K.L. Lee K.M. Villereal M. Rosner M.R. J. Biol. Chem. 1992; 267: 19876-19883Abstract Full Text PDF PubMed Google Scholar) (Fig.3 A, lane 4) clearly attenuated GnRHa-induced ERK activation, indicating that intracellular Ca2+ is also required for GnRHa-induced ERK activation. On the other
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